My research program is fueled by the desire to elucidate the biology of understudied fungal pathogens through the development and application of molecular and genetic tools. As a graduate student I used molecular genetics to identify a key regulator of asymmetric cell fate in Saccharomyces cerevisiae. After finishing graduate school, I launched an independent research program instead of pursuing a traditional postdoctoral fellowship. By that time I had developed great respect for how genetics can uncover the factors that drive biological processes. I decided to apply these approaches to the regulatory circuits that govern the underlying biology of the fungal pathogen Histoplasma capsulatum. I was drawn to this organism because of its fascinating behaviors: it grows robustly in the diverse environments of human and soil, and temperature plays a key role in triggering the establishment and maintenance of a growth program specialized for each milieu. In the soil, cells grow in a filamentous form that also produces vegetative spores. When spores or fragments of filaments are aerosolized and inhaled by the host, they convert into a budding yeast form that replicates vigorously within macrophages. Despite the clinical notoriety enjoyed by H. capsulatum, little is known about how it is able to switch morphology in response to temperature, and how it manipulates the innate immune system so effectively.
My research is driven by two key questions. First, how do cells sense temperature and make a developmental switch from the soil to the host program? I focus on temperature because it is a sufficient signal to recapitulate the morphologic switch between Histoplasma filaments (the soil form) and yeast (the host form) in culture. This question is critical to understanding the basic biology of Histoplasma as well as that of a number of closely related fungi, such as Blastomyces, Coccidioides, and Paracoccidioides, each of which is a ubiquitous pathogen of immunocompetent hosts in endemic areas. One of the fascinating questions about these environmental fungi is how regulatory circuits have evolved to link morphology and virulence programs with growth at host temperature. Studying the regulation of Histoplasma development by temperature will also be an entry point to broader studies of host-fungal interactions, since this research will define critical developmental changes that promote the expression of virulence traits. It will also delineate molecular landmarks that will allow us to stage the interactions of the fungus with host cells. Finally, it is likely that a potentially unique molecular mechanism underlies the ability of cells to sense an ephemeral property, such as the temperature of their environment.
I hypothesize that a particular molecule (e.g., a lipid, transcript, or protein) is temperature sensitive, such that it adopts a unique confirmation under different environmental conditions. To identify such a factor, my lab performed one of the first large-scale screens in H. capsulatum. We obtained a number of mutants that are trapped in the filamentous form independent of temperature and used these strains to identify genes that are required for yeast-phase growth (RYP genes) at 37oC. Currently our main focus is to understand the molecular functions of these genes and to determine how the activities of their gene products are regulated by temperature.
Our second key question asks how H. capsulatum defies the innate immune response to take up residence, often permanent, in immunocompetent hosts. The past 10 years have witnessed an exponential increase in our understanding of the innate immune response to microbes, and yet, in the case of fungi, our insight is rudimentary at best. Our studies explore the molecular communication at the host-pathogen interface between H. capsulatum and the macrophage, and will likely influence the design of new approaches to combat intracellular pathogens in general.
My goal is to use Histoplasma as a model to explore the confrontation between the innate immune system and fungal pathogens. Although the interaction between the host and a number of bacterial intracellular pathogens has been well characterized, we understand little about how eukaryotic pathogens manipulate the eukaryotic host cell. H. capsulatum displays extremely robust macrophage colonization, so it is one of the best fungal candidates to probe the Achilles' heel of these powerful innate immune cells and determine novel mechanisms of virulence that have evolved in eukaryotic pathogens.
Intracellular pathogens use their host cells as a safe place to reside and replicate. Nonetheless, the need to disseminate can necessitate escape from the host cell under the right conditions. In the case of Histoplasma, macrophages become choked with large numbers of yeast cells, and lysis of the host cell ensues. Whether the host cell simply bursts due to escalating numbers of yeast cells, or whether a Histoplasma factor accumulates and provokes lysis, is unknown.
We have taken genetic and genomic approaches to understand the interplay between host and pathogen, and these two approaches have recently dovetailed to suggest that Histoplasma actively triggers host cell lysis. By performing a large-scale genetic screen, we identified 27 Histoplasma mutants that fail to colonize macrophages. A large fraction of these mutants fail to grow well in macrophages; future studies will further our understanding of fungal genes that are required for survival in immune cells. To our surprise, 4 of the 27 mutants did not display a growth defect in macrophages. Instead, these mutants reached even higher numbers of yeast cells per macrophage than wild-type Histoplasma. In contrast to wild type, however, infection with these mutants did not result in macrophage lysis, suggesting that the mutants are deficient in a factor or factors that promote host cell death. Current studies indicate that at least three of the mutants are deficient in the same secreted factor, and we are pursuing the role of this factor in host cell lysis.
We have also used mouse microarrays to define the transcriptional response of macrophages to infection with Histoplasma. By comparing these data with the transcriptional response of macrophages to intracellular bacterial pathogens, we discovered a small cluster of genes that is induced only by Histoplasma. This Histoplasma-specific cluster is not induced if we infect macrophages with UV-treated Histoplasma, suggesting that its induction requires the presence of replicating yeast cells. The cluster contains transcripts associated with stress response and modulation of cell death. The observation that this cluster fails to be induced during infection with the Histoplasma mutants that grow robustly within macrophages but fail to lyse them is significant. This provocative result suggests that induction of the cluster by Histoplasma correlates with the ability of the fungus to trigger macrophage lysis. We are pursuing the hypothesis that induction of these host genes by Histoplasma either initiates a lytic program in the macrophage or represents the host response to a lytic program. Thus we are now poised to decipher the molecular dialogue between host and pathogen during host cell colonization. These studies are vital to our understanding of the evolution of virulence determinants in eukaryotes.
Grants from the National Institutes of Health, the Burroughs Wellcome Fund, the Ellison Medical Foundation, the Sandler Center for Basic Research, and the American Cancer Society provided partial support for these projects.
As of May 30, 2012